中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (8): 557-560.

• 论著 • 上一篇    下一篇

氨基酮戊酸光动力疗法对浮游表皮葡萄球菌抑制作用的体外研究

李欣1,王秀丽2,王宏伟2,张玲琳2   

  1. 1. 徐州市中心医院皮肤科
    2. 上海市皮肤病医院
  • 收稿日期:2011-09-01 修回日期:2011-10-13 出版日期:2012-08-15 发布日期:2012-08-01
  • 通讯作者: 王秀丽 E-mail:xlwang@yahoo.cn

Inhibition of Staphylococcus epidermidis planktonic cells by 5-aminolevulinic acid-based photodynamic therapy

  • Received:2011-09-01 Revised:2011-10-13 Online:2012-08-15 Published:2012-08-01

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摘要：

目的 观察氨基酮戊酸光动力疗法（ALA-PDT）对浮游表皮葡萄球菌的影响，探讨ALA最适浓度和最佳孵育时间。 方法 实验分3组，第1组，50 mmol/L ALA与细菌37 ℃避光孵育3、5、8、12、16、18、20、24 h；第2组，不同浓度ALA（10、20、30、40、50 mmol/L）与细菌37 ℃避光孵育16 h；第3组，单纯胰蛋白胨大豆肉汤培养基（TSB）（不加ALA）与细菌37 ℃避光孵育24 h。3个组均通过激光共聚焦显微镜（CLSM）检测不同时间点原卟啉Ⅸ（PpⅨ）的荧光强度并做定量分析。同时对第1组进行不同剂量（30、50、70、90、100 J/cm2）红光照射，第2组用100 J/cm2红光照射，第3组不照光，同时设不同剂量红光照射对照组。采用菌落计数法分析ALA-PDT对浮游表皮葡萄球菌的影响。结果 第1组，CLSM下均观察到砖红色荧光，荧光强度随着孵育时间的延长而逐渐增强，孵育16、18、20、24 h的荧光强度明显高于3、5、8、12 h（P < 0.05）；第2组，荧光强度随着ALA浓度的增加而增强，50 mmol/L ALA组荧光强度明显高于10、20、30、40 mmol/L ALA组（P < 0.05）；第3组，未见砖红色荧光。前两组经红光照射后发现，随着ALA浓度和光剂量的增加，存活的细菌数逐渐减少，当ALA为50 mmol/L、光剂量为100 J/cm2时，细菌生长受到抑制。结论 ALA-PDT对浮游表皮葡萄球菌有明显抑制作用，最适治疗参数为50 mmol/L ALA、孵育16 h，光照剂量100 J/cm2。

关键词: 浮游菌

Abstract:

Objective To observe the effect of 5-aminolevulinic acid-based photodynamic therapy （ALA-PDT） on S. epidermidis planktonic cells, and to determine the optimal concentration and incubation time of ALA. Methods Some S. epidermidis planktonic cells were divided into 3 groups to be incubated with ALA at 50 mmol/L for different durations （3, 5, 8, 12, 16, 18, 20 and 24 hours） at 37 ℃ in dark room （group 1）, with ALA at various concentrations （10, 20, 30, 40 and 50 mmol/L） for 16 hours at 37 ℃ in dark room （group 2）, and with fresh trypticase soya broth （TSB） solution for 24 hours at 37 ℃ in dark room （group 3） , respectively. Confocal laser scanning microscopy （CLSM） was used to measure the fluorescence intensity of protoporphyrin IX （PpIX） in bacterial suspensions at different time points. Additionally, some S. epidermidis planktonic cells in group 1 were irradiated with red light at 30, 50, 70, 90 and 100 J/cm2 after incubation with ALA at 50 mmol/L for 16 hours, some in group 2 were irradiated with red light at 100 J/cm2 after incubation with ALA for 16 hours, those cells in group 3 received no irradiation （blank control group）, and some S. epidermidis planktonic cells receiving only irradiation and no pretreatment with ALA served as the laser control group; subsequently, the bacterial suspension was inoculated onto trypticase soy agar （TSA） followed by the calculation of colony forming units （CFUs） of S. epidermidis. Results Brick red fluorescence was observed by CLSM in S. epidermidis planktonic cells in group 1 and 2, but not in those in group 3, and the fluorescence intensity was enhanced with the increase in incubation time and concentration of ALA. In detail, the fluorescence intensity was significantly higher in planktonic S. epidermidis cells after 16-, 18-, 20- and 24-hour incubation than in those after 3-, 5-, 8- and 12-hour incubation with ALA at 50 mmol/L （all P < 0.05）, and higher in those incubated with ALA at 50 mmol/L than in those with ALA lower than 50 mmol/L （all P < 0.05）. After irradiation, the number of surviving S. epidermidis planktonic cells declined with the increase in ALA concentration and red light doses, statistically lower in the group 1 and 2 than in the blank control group （both P < 0.05）, but similar between the blank control and laser control group （P > 0.05）. The growth of planktonic cells was inhibited after incubation with ALA at 50 mmol/L and irradiation with red light at 100 J/cm2. Conclusions ALA-PDT shows a marked inhibitive effect on S. epidermidis planktonic cells, with the optimal working concentration of ALA being 50 mmol/L, incubation time 16 hours, and dose of red light 100 J/cm2.

Key words: Planktonic cells